The present invention, in some embodiments thereof, relates to the use of plant cells expressing a TNFalpha polypeptide inhibitor in therapy.
Tumor necrosis factor alpha (TNFα) is an important, pro-inflammatory cytokine mediating the regulation of diverse inflammatory, infectious and immune-related processes and diseases, TNFα being considered the most important mediator responsible for inflammatory pathology.
TNF-alpha is a 17 kD molecular weight protein, initially synthesized as a transmembrane protein arranged in stable trimers, then cleaved by metalloprotease-TNF alpha converting enzyme (TACE) to form the homotrimeric soluble TNF (sTNF) which engages to its cognate receptors (TNFRI, p55 and TNFRII, p75), expressed ubiquitously. The ubiquitous TNF receptors provides the basis for the wide variety of TNF-alpha mediated cellular responses.
TNF-alpha induces a wide variety of cellular responses, many of which result in deleterious consequences, such as cachexia (loss of fat and whole body protein depletion, leading to anorexia, common in cancer and AIDS patients) and septic shock. Elevated secretion of TNF-alpha has been implicated in a variety of human diseases including diabetes, allograft rejection, sepsis, inflammatory bowel diseases, osteoporosis, in many autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, psoriatic arthritis, hypersensitivity, immune complex diseases, and even in malaria, cancer and lung fibrosis.
The biological effect of TNFα is mediated by the two distinct receptors. TNF-alpha receptors, when shed from mononuclear cells, lower the TNF-alpha levels by “mopping up” and acting as natural inhibitors Neutralization of TNFα by specific antibodies and decoy receptors has become a common strategy for regulation of TNFα mediated toxicity.
To date, five protein-based TNFα antagonists have been approved by the US FDA for clinical use: Cimzia (Certolizumab pegol), a TNFmAb Fab′ fragment—PEG conjugate; Remicade (Infliximab), a TNF rmAB; Humira (Adalimumab, a TNF rmAB, Simponi™ (Golimumab), an anti-TNF and etanercept, a fusion protein of soluble 75 kDa TNFα receptors fused to the Fc fragment of human IgG (registered as Enbrel™).
Etanercept is indicated for rheumatoid arthritis (RA) and other arthritic indications such as juvenile idiopathic arthritis (JIA), psoriasis and Ankylosing Spondylitis (AS). Rheumatoid arthritis (RA) is a chronic disease that affects approximately five million people World Wide. Nearly 500,000 patients worldwide across indications are treated with Enbrel. Enbrel sales in 2010 were 7.8 billion dollars and the total anti-TNF market amounted to 24.04 Billion dollars. Clinical trials of Enbrel therapy, current or completed, include such diverse indications as adult respiratory distress syndrome, pemphigus, Alzheimer's disease, Behcet's syndrome, HIV, myocardial infarct, knee joint synovitis, lupus nephritis, lichen planus, systemic amyloidosis, sciatica, vitiligo, chronic fatigue syndrome, anorexia, TMJ, asthma, bronchitis, diabetes, myelodysplastic disease and others.
Biopharmaceuticals typically pose a number of challenges, however, that drug developers must overcome in order to successfully develop these compounds into safe and effective therapeutics. For example, proteins and peptides tend to be destroyed by proteolytic enzymes or, in the case of the higher molecular weight proteins, may generate neutralizing antibodies. Moreover, large complex molecules can exhibit low solubility or poor stability, leading to short shelf lives. As a result, biopharmaceutical therapeutics often quickly lose their effectiveness or require frequent dosing. These factors impact not only cost of therapy, but also patient acceptance and compliance, thus affecting their therapeutic efficacy.
Oral Administration:
The most common mode of protein and peptide-based administration is by invasive methods of drug delivery, such as injections and infusions. Although these are the primary modes for administering macromolecular drugs for systemic diseases, they are also the least desirable for patients and practitioners. The obvious downside of this delivery method is patient acceptance and compliance, limiting most macromolecule development to indications in which the need to use invasive administration routes are not outweighed by associated expenses or inconvenience. As a simple, non-invasive method for systemically delivering drugs, oral administration provides many advantages: ease and convenience of use, access to extensive volume of absorptive surface, high degree of vascularization, relatively lengthy retention time, natural disposal of inactive, non-metabolized ingredients, and more.
Nonetheless, investigations of oral administration of macromolecular pharmaceuticals have not indicated satisfactory levels of efficiency to match the potential of this route. Some of the obstacles are difficulties of ingestion of pills and other solid formulations, lability of biologically active macromolecules in the GI tract, concentration of the biologically active agents at the mucosa, and permeability of GI membranes to biologically active macromolecules.
The oral route of administration of biologically active substances is complicated by both high acidity and enzymatic degradation in the stomach, which can inactivate or destroy biologically active macromolecules before they reach their intended target tissue. Further, effective concentrations of a biologically active macromolecule are difficult to achieve in the large volumes encountered in the GI tract. Thus, to be effective, most drugs must be protected from absorption and/or the environment in the upper GI tract, and then be abruptly released into the intestine or colon. Various strategies are being developed in the pharmaceutical industry to overcome the problems associated with oral or enteral administration of therapeutic macromolecules such as proteins. These strategies include covalent linkage with a carrier, coatings and formulations (pH sensitive coatings, polymers and multi-layered coatings, encapsulation, timed release formulations, bioadhesives systems, osmotic controlled delivery systems, etc) designed to slow or prevent release of active ingredients in harsh conditions such as the stomach and upper GI tract. However, preparation of biologically active agents in such formulations requires complex and costly processes. Also employed are mucosal adhesives and penetration enhancers (salicylates, lipid-bile salt-mixed micelles, glycerides, acylcarnitines, etc) for increasing uptake at the mucosa. However, some of these can cause serious local toxicity problems, such as local irritation, abrasion of the epithelial layer and inflammation of tissue. Other strategies to improve oral delivery include mixing the biologically active agent with protease inhibitors, such as aprotinin, soybean trypsin inhibitor, and amastatin; however, enzyme inhibitors are not selective, and also inhibit endogenous macromolecules, causing undesirable side effects. Thus, present methods of oral administration of biologically active biopharmaceuticals cannot ensure efficient delivery of desired biological activity at the target tissue. Attempts at orally administering TNFR2:Fc (Enbrel) have failed to due to the high acidity and enzymatic degradation in the stomach that inactivates or destroys the molecule before reaching the circulation. Elaborate, complicated mechanisms, including devices for automatic parenteral administration have evolved to ensure compliance with dosage regimens.
Additional background art includes: U.S. Pat. No. 7,915,225 to Finck et al, U.S. patent application Ser. Nos. 13/021,545 and 10/853,479 to Finck et al, U.S. patent application Ser. No. 11/906,600 to Li et al, U.S. patent application Ser. No. 10/115,625 to Warren et al and U.S. patent application Ser. No. 11/784,538 to Gombotz et al.